Unraveling iron oxides as abiotic catalysts of organic phosphorus recycling in soil and sediment matrices

In biogeochemical phosphorus cycling, iron oxide minerals are acknowledged as strong adsorbents of inorganic and organic phosphorus. Dephosphorylation of organic phosphorus is attributed only to biological processes, but iron oxides could also catalyze this reaction. Evidence of this abiotic catalysis has relied on monitoring products in solution, thereby ignoring iron oxides as both catalysts and adsorbents. Here we apply high-resolution mass spectrometry and X-ray absorption spectroscopy to characterize dissolved and particulate phosphorus species, respectively. In soil and sediment samples reacted with ribonucleotides, we uncover the abiotic production of particulate inorganic phosphate associated specifically with iron oxides. Reactions of various organic phosphorus compounds with the different minerals identified in the environmental samples reveal up to ten-fold greater catalytic reactivities with iron oxides than with silicate and aluminosilicate minerals. Importantly, accounting for inorganic phosphate both in solution and mineral-bound, the dephosphorylation rates of iron oxides were within reported enzymatic rates in soils. Our findings thus imply a missing abiotic axiom for organic phosphorus mineralization in phosphorus cycling.

(5) What are the parameters in your soil and sediment samples' porewater?Local temperature?You set up pH 7 and 0.1 M NaNO3 + 0.01 M NaHCO3 to perform experiments.Why not use the porewater condifions to do experiments?Which might be closer to realisfic reacfion condifions and show the true catalyfic reacfivity/rates of Fe oxides in organic P mineralizafion.(6) You used XRD to do mineralogical idenfificafion.How do you know amorphous phase contents and how do they affect your conclusions?(7) Figure S6 showed the similar XANES spectra for both mineral-Porgnic and mineral-Pi?What is the confidenfiality of using P K-edge XANES to analyze surface organic and inorganic P? Overall, the manuscript is of high quality.I assume that considering these concerns/comments might further improve your manuscript quality and consolidate your findings about this new and interesfing role of Fe oxides in P biogeochemical cycles in the sub-surface Earth.Reviewer #2 (Remarks to the Author):

References
I have concluded that the research described is sufficient quality and potenfial impact to be accepted for publicafion in Nature Communicafions, with some minor revision (see below).The manuscript is very well wriften and presented, and the findings of this study clearly demonstrated that significant mineralizafion of model organic phosphorus compounds occurred on the surface of iron oxide, which represents a potenfially important process that contributes to phosphorus transformafions and bioavailability in sediments and soils.I only have one significant comment/request to make on the manuscript.I am a soil scienfist with very liftle experience or experfise in sediment phosphorus dynamics.With specific regard to the dynamics of organic phosphorus in soil, I was surprised to note the complete absence of any menfion or considerafion of the role and funcfion of "iron oxide surface mineralzafion" in the cycling and bioavailability of inositol phosphates.Inositol phosphates (principally inositol hexaphosphate [commonly referred to as "phytate"]) are widely acknowledged to be important consfituenst of soil organic phosphorus, and have been shown to be strongly adsorbed on soil colloid surfaces.I would request that the authors consider including some discussion of the potenfial role of iron oxide surface mineralizafion in the fate and bioavailability of inositol phosphates in soil.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): NCOMMS-23-32052: Iron Oxides as Catalytic Traps in Organic Phosphorus Mineralization Basinski et al., showed and quantified a new and crucial role of iron oxides as an abiotic mineral-enzyme in organic P remineralization.Some critical and interesting roles of environmental Fe and Mn oxides are found in recent research, such as iron-and manganese as catalysis to cause the transformation of simple organic molecules into complex macromolecules (Moore et al., Nature, 2023).This manuscript is also an interesting paper to include the quantitative analyses of solution and surface P species transformation into soil and sediment incubation.The author then found that the Fe oxides exhibited up to great high catalytic reactivity (or dephosphorylation activity) for ribonucleotide phosphate mineralization in soils and sediments.Finally, the writings and figure plots are well organized to show the findings and results.However, there still are many concerns that have to be processed before the acceptance of this manuscript.Thus, the current version cannot be accepted by Nature Communications and I suggested a major revision.
Response: Thank you for acknowledging the new underexplored yet critical role of iron oxides with enzyme-like characteristics in the phosphorus cycle.We appreciate the highlight of our well-written and well-organized by the reviewers.Below we provide details on how we have invested in addressing all the concerns and feedbacks by the reviewer.Notably, we were able to secure additional beamtime at the Australin Synchrotron on short notice to conduct to analyze samples stemming from the additional experiments the author requested with other organic P types.This would have been an impossible task for us to achieve due to the shutdown of the Stanford synchrotron.We are grateful to our diverse team of co-authors that includes beamline scientists from different synchrotron facilities.Response: We agree with the reviewer that phosphomonoesters (with C-O-P) are found to be the forms of Porg mostly detected in environmental matrices.In our original manuscript, we did present data with both AMP (a phosphomonoester) and ATP reacted with the different Fe oxides, quartz, aluminosilicates.We used the data with the two different ribonucleotides to make a statement that the Fe oxide reactivity would be dependent on the Porg types because while goethite and hematite has more reactivity towards ATP than AMP, ferrihydrite had more catalytic reactivity for AMP than for ATP.However, as pointed out by the reviewer, we acknowledge there are different forms of Porg compounds with phosphomonoester bonds.To address the concern by the reviewer that we need to consider how different phosphomonesters may react with the Fe oxides, we conducted additional experiments with a sugar phosphate (glucose-6-phosphate) and phytate (the primary storage in plants), to add to our data with AMP.These new data are illustrated in Figures 2d and 2e.We expanded the text to discuss the new results along with the discussion of AMP (see Lines 212-251).It is important to acknowledge that Porg with phosphoanhydride bonds (P-O-P) are ubiquitous in plant and microbial metabolism and biomass.Therefore, the persistent detection of phosphomonoesters may be indicative of both low reactivity of phosphomonoesters and the accumulation of dephosphorylation products from multiphosphorylated Porg compounds containing phosphoanhydride bonds.In fact, in agreement with previous enzymatic dephosphorylation data, we found two of Fe oxides had relatively lower reactivity for phosphomonoesters had lower reactivity compared to ATP, but, contrary to the enzyme results, AMP reactivity was nearly 20-fold greater on ferrihydrite relative to ATP reactivity on ferrihydrite.We added to the discussion section to highlight these findings (see Lines 407-424).This remarkable difference in mineral reactivity was only captured by surface characterization by P-XANES.We note that the publication by Wan et.al. 2022 only monitored the generation of Pi in solution.With regards to experiments with the natural samples, we only performed reactions with ATP due to insufficient amounts of natural samples that precluded us from conducting experiments with both ATP and the entire set of phosphomonoesters.
In contrast to the ATP reactions, all the Pi derived from the reacted AMP was retained as particulate Pi while aqueous Pi was absent, a significant finding that was made possible here due to the application of the XANES technique (Fig. 2c).On the one hand, the catalytic reactivity of ferrihydrite was higher for AMP than for ATP, as reflected by the 20-fold increase in particulate Pi fraction (p < 0.001) (Fig. 2a and 2c).On the other hand, the catalytic reactivity of goethite was less for AMP than for ATP as characterized by a 3-fold decrease in particulate Pi fraction (p < 0.01) accompanied by no change in the particulate Porg fraction (p = 0.29) and no measured solution Pi; hematite did not display any adsorption or catalytic reactivity towards AMP (Fig. 2a and 2c).Interestingly, in our aforementioned experiments with ATP and goethite, 20-24% of the initial ATP-P remained as AMP-P in solution and no ADP was detected after nearly all the ATP reacted with goethite was transformed or adsorbed (SI, Fig. S10).Thus, this accumulation of AMP in the ATPgoethite experiment can be explained by the lower reactivity of goethite for AMP relative to ATP (Fig. 2c; SI, Fig. S10).When ATP was reacted with hematite, we measured a conversion of 17-23% of the initial ATP-P to ADP-P and 5-7% to AMP-P, consistent with lower reactivity for both ADP and AMP compared to ATP (SI, Fig. S10).Notably, the silicate and aluminosilicate minerals did not exhibit any adsorption or catalytic reactivity towards AMP (Fig. 2c).
In contrast to AMP, all the silicate and aluminosilicate minerals adsorbed phytate (from 5% to 50% of the total reacted phytate-P) and, rather than an Fe oxide, the clay illite adsorbed the most phytate (Fig. 2d).However, similar to the results with AMP and ATP, catalytic reactivity towards phytate was only obtained with Fe oxides, specifically hematite and goethite (Fig. 2d).Relative to controls, phytate-derived particulate Pi was higher by 8 -14% (p < 0.01) with hematite and by 24 -32% with goethite (p < 0.001); the particulate Pi with ferrihydrite, however, corresponded to the adsorption of solution Pi in the control experiment (Fig. 2d).For G6P, some G6P adsorption was observed with two of the aluminosilicates (mica and kaolinite) and two of the Fe oxides (ferrihydrite and goethite) (Fig. 2e).But, as with phytate, the most significant adsorption was with ferrihydrite, with 47 -78% of the reacted G6P found as particulate Porg (Fig. 2e).None of the investigated minerals catalyzed G6P dephosphorylation (Fig. 2e).
In sum, our data revealed that the silicates and aluminosilicates either had minimal to no catalytic reactivity or exhibited some extent of adsorption reactivity.Only the Fe oxides were found to catalyze the dephosphorylation of both phosphomonoester and phosphoanhydride-bearing compounds.Importantly, the Fe oxide-catalyzed reactions seemed to be dependent on both the mineral surface chemistry and the type of Porg species.As proposed previously 14 , we expect the differences in reactivity may stem from the binding conformations of different Porg on the mineral surface.

Lines 407-424:
While phosphoanhydride-containing compounds are not commonly detected in soil and sediment systems 34,35 , phosphoanhydride bonds are ubiquitous in plant and microbial metabolites, including ATP as the major P content in many microbes 4,52 .We posit that the persistent detection of phosphomonoesters in environmental matrices may be indicative of both low reactivity of enzymes and minerals towards phosphomonoesters and the accumulation of remnants of dephosphorylated Porg compounds containing phosphoanhydride bonds.Here we show that the Fe oxide-catalyzed reaction for a Porg compound containing both phosphoanhydride and phosphoester bonds was retained, even in heterogeneous mixtures with quartz or clays.We also found accumulation of a monophosphorylated ribonucleotide after Fe oxide-catalyzed reactions with a triphosphorylated ribonucleotide.A study on enzymatic dephosphorylation 53 reported higher reactivity, by approximately 2 orders of magnitude, for multiphosphorylated compounds than for phosphomonoester compounds.Goethite and hematite, both of which represented the most reactive Fe-oxides, had minimal reactivity towards phosphomonoester Porg compounds.This lack of significant dephosphorylation of phytate and a sugar phosphate by the Fe oxide minerals may contribute to the persistence of these phosphoester Porg types in environmental matrices.However, dephosphorylation of a monophosphorylated ribonucleotide by ferrihydrite, was nearly 20-fold greater to that for the triphosphorylated ribonucleotide.Therefore, both the Fe oxide type and the Porg chemistry need to be considered when predicting the extent of the catalytic fate of Porg in different environmental matrices.

Comment 2:
How about manganese oxides in organic P mineralization in soils and sediments since Mn oxides also show high reactivity to P transformation (Baldwin et  Response: We agree with the reviewer that Mn oxides are known to be reactive towards organic P compounds, as we had pointed out in the Introduction of our original manuscript (see Lines 57 and lines 63).However, the scope of our project is on Fe oxides, especially since there was no quantifiable Mn amount (XRF data) or identifiable Mn-bearing minerals (XRD data) in our natural soil and sediment samples (See lines 136-137).We also expanded our discussion to point to the reason why reactivity with Mn oxides was not considered in our experiments but also highlight their possible significance in marine sediments.(See lines 430-439).
Lines 136-137: No manganese-bearing minerals were included in the mineral composition analysis because neither environmental sample exhibited a quantifiable amount of manganese (SI, Fig. S1).
Lines 430-439: Current models of P cycling 1,3,56 , which include enzymes for Porg dephosphorylation and minerals for Porg adsorption, do not account for reported catalytic dephosphorylation of Porg by Fe oxides and Mn oxides 3,14,16 .We only consider Fe oxides in this research due to no quantifiable Mn in our forest soil and lake sediment samples, but there can be approximately, on a per-mass basis, 10:1 Fe oxides:Mn oxides in marine sediments 57,58 .Our quantitative findings imply an important and yet unaccounted dual catalytic and adsorbent role of Fe oxides which warrant consideration alongside biologically mediated processes in the P cycle.Our proposed redefined role of Fe oxides as important catalytic players, if confirmed to be a widespread phenomenon with quantitative significance, would have important implications regarding the addition of an abiotic axiom at the Porg-Pi nexus in the biogeochemical P cycling.

Comment 3:
You should include the XRF method of P measurement (Figures 2 c-d) in the method section.Did you XRF in the SSRL 2-3 (Energy: 4.9-23 keV)?Could SSRL 2-3 measure P mapping?Why not use SSRL 14-3?

Response:
As the reviewer pointed out, we did perform the XRF mapping on SSRL's beamline 2-3 and have now included the methodology of those P measurements (See Lines 517-521).The P mapping acquired at the 2-3 beamline was solely used for P and Fe correlation analysis.We note that, while the 2-3 beamline at SSRL can perform P mapping, it does not have the same sensitivity as the 14-3 beamline.We also ran the samples with the intention of P mapping at the 14-3 beamline, but we unfortunately do not have the data due to an issue with the memory during data collection.However, because we only used the data from the 2-3 beamline for correlation analysis, the lower sensitivity at that beamline was not an issue.

Lines 517-521:
We also collected μ-XRF maps on this beamline using 3 different energies across the P adsorption edge: 2148, 2152.3, and 2152.5 eV.Because the 2-3 beamline is not as sensitive as the 14-3 beamline, the P mapping was solely used to identify P hotspots in the samples and confirm P localization with Fe.Calibration for P measurements was performed by setting the maximum of the first derivative of the XANES spectrum of GaP to 2152.0 eV.

Comment 4:
Line 235-236 Autoclave sterilization?Generally, increasing temperature will significantly change the mineralogy of soils and sediments and might increase the contents of crystalline hematite and goethite?Could you please add the new Fe XANES to compare the mineralogical change of Fe oxides in your soil and sediment samples due to autoclave treatment?Maybe, you also need to consider how this sterilization way affects your findings and conclusions.
Response: As the reviewer pointed out, autoclaving of samples containing Fe oxides would likely change the mineralogy of these minerals.For that reason, our natural samples were not autoclaved and biological dephosphorylation was assumed to be minimal due to long-term storage and low carbon loading.We apologize for the typo and have edited the section that included this information (see Lines 278-286).
Lines 278-286: Biologically-mediated Porg dephosphorylation was not expected to be significant in our natural samples due to long-term storage (~4 years) of both samples and their low carbon loading (<0.2% g C g -1 soil) particularly for the soil sample.Nevertheless, we tested the possibility of residual microbial or enzymatic reactions in the natural samples by performing experiments with an antimicrobial agent or an enzyme denaturing agent, respectively (SI, Fig. S12).We determined that these biotic reactions accounted only for 0 -5% and 23 -26% of the total reactivity in the sediment and soil samples, respectively (Fig. 3a and 2c).Taken collectively, our findings bring attention to the occurrence of a pool of abiotically generated particulate Pi from mineral-mediated Porg transformation that has been hitherto unaccounted for in environmental matrices.Response: The pH of the porewater at the Calhoun Critical Zone Observatory soil was determined to be 5.8 at the time of excavation, however the reported pH at this site has ranged from 4.5 to 6.2 (Richter et.al. 1994; Chen et.al. 2020).The mean annual temperature at the soil sampling site is 16 °C with a range of 5 °C to 25 °C (Richter et.al. 1994).For the Missisquoi Bay sediment sample, the porewater was determined to be at a temperature of 24 °C with a circumneutral pH (Smith et.al. 2011 and Cai et.al. 2010).We have included this information in Lines 468-473.Rather than using reaction conditions that directly mirrored those of the porewater conditions of the natural systems, we chose to use the standardized conditions of pH 7 and background electrolyte 0.1 M NaNO3 buffered with 0.01 M NaHCO3 at 25 °C to be able to compare directly the reactivities of the different minerals present across the two different systems.Please see lines 548-553 in the main text for the inclusion of this reasoning.

Lines 468-473:
The pH of the porewater at the Calhoun Critical Zone Observatory soil was determined to be 5.8 at the time of excavation, however the pH has been reported previously to range between and 4.5 to 6.2 57,58 .The mean annual temperature at the soil sampling site is 289 K (or 16 ºC), ranging temperatures found to range between 278 K (5 ºC) and 298 K (25 ºC) 57 .For the Missisquoi Bay sediment sample, the porewater pH was determined to be circumneutral with a temperature of 297K (24.0 ºC) 59,60 .
Lines 548-553: Here, to compare directly the reactivities of the different minerals present across the two different natural samples, we chose to use the standardized conditions of a reaction solution at pH 7.0, ionic strength set by 0.1 M NaNO3, and buffered by 0.01M NaHCO3 at 298K (25 ºC).We acknowledge that the actual porewater conditions in the natural samples, as highlighted above under detailed descriptions of sample characteristics, would be different from the standardized experimental conditions.

You used XRD to do mineralogical identification. How do you know amorphous phase contents and how do they affect your conclusions?
Response: We acknowledge that our characterization method focused on the crystalline phases via XRD and that only the amorphous phases were that of Fe phases characterized via XANES.The scope of our technical analysis did not account for possible amorphous silicate and aluminosilicate phases.However, according to our data, these silicate-bearing minerals all had low catalytic and adsorption reactivities compared to the Fe oxides for our representative Porg.Therefore, we do not expect the presence of amorphous phases would affect the fate of the Porg reactant.The main text has been modified (See Lines 151 -157) to express this caveat.

Lines 151-157:
Moreover, our XRD and Fe XANES data would not account for the possible presence of lowcrystallinity silicate or aluminosilicate phases in the natural samples.However, as will be discussed in the next section, there was minimal to no catalytic reactivity of silicate or aluminosilicate silicates towards the different Porg compounds (Fig. 2).Consistently, based on our analysis, we found that about 80% (or more) on a per-mass basis of both sediment and soil samples was comprised of silicate minerals of different types (quartz, micas, feldspars, clays), and less than 20% constituted the Fe oxide fraction (Fig. 1c).

Comment 7:
Figure S6 showed the similar XANES spectra for both mineral-Porgnic and mineral-Pi?What is the confidentiality of using P K-edge XANES to analyze surface organic and inorganic P? Overall, the manuscript is of high quality.I assume that considering these concerns/comments might further improve your manuscript quality and consolidate your findings about this new and interesting role of Fe oxides in P biogeochemical cycles in the sub-surface Earth.
Response: Recent studies performed by Prietzel et.al., 2016, Klein et.al., 2019, and Eusterhues  et.al., 2023, have shown that P K-edge spectra LCF can be used to differentiate the difference between Fe-Porg and Fe-Pi.This is due to the analysis of the pre-edge feature, shifting of the white line, and post-edge features.As pointed out by the reviewer, it is difficult to discern differences in the Pi and Porg reference spectra and reaction spectra in the original S6 figure.We have included an inset that focuses on the white line region and the post-edge region so this distinction is more apparent.Please see the new Figure S6.accepted for publication in Nature Communications, with some minor revision (see below).The manuscript is very well written and presented, and the findings of this study clearly demonstrated that significant mineralization of model organic phosphorus compounds occurred on the surface of iron oxide, which represents a potentially important process that contributes to phosphorus transformations and bioavailability in sediments and soils.

Response:
We thank the reviewer for their kind words regarding our effort to present a wellwritten manuscript and acknowledging the important contribution of our research findings.

Main comments:
Comment 1: I only have one significant comment/request to make on the manuscript.I am a soil scientist with very little experience or expertise in sediment phosphorus dynamics.With specific regard to the dynamics of organic phosphorus in soil, I was surprised to note the complete absence of any mention or consideration of the role and function of "iron oxide surface mineralzation" in the cycling and bioavailability of inositol phosphates.Inositol phosphates (principally inositol hexaphosphate [commonly referred to as "phytate"]) are widely acknowledged to be important constituenst of soil organic phosphorus, and have been shown to be strongly adsorbed on soil colloid surfaces.I would request that the authors consider including some discussion of the potential role of iron oxide surface mineralization in the fate and bioavailability of inositol phosphates in soil.

Response:
We agree with the reviewer that inositol phosphate or phytate is an important component of soil Porg reservoirs.We conducted additional experiments involving reactions of phytate with the three Fe oxides (ferrihydrite, hematite, and goethite), quartz, and the aluminosilicates with inositol hexaphosphate or phytate.We also performed experiments with the sugar phosphate G6P, another important Porg in plant and microbial systems.The results can be found in new Figure 2d, and 2e and the accompanying discussion in see Lines 212-251.Our results show that the catalytic reactivity of Fe oxides (specifically goethite and hematite) towards phytate was significantly lower than with ATP, but the Fe oxide was the only mineral demonstrating catalytic dephosphorylation of phytate compared to the other silicate and aluminosilicate minerals.
In contrast to the ATP reactions, all the Pi derived from the reacted AMP was retained as particulate Pi while aqueous Pi was absent, a significant finding that was made possible here due to the application of the XANES technique (Fig. 2c).On the one hand, the catalytic reactivity of ferrihydrite was higher for AMP than for ATP, as reflected by the 20-fold increase in particulate Pi fraction (p < 0.001) (Fig. 2a and 2c).On the other hand, the catalytic reactivity of goethite was less for AMP than for ATP as characterized by a 3-fold decrease in particulate Pi fraction (p < 0.01) accompanied by no change in the particulate Porg fraction (p = 0.29) and no measured solution Pi; hematite did not display any adsorption or catalytic reactivity towards AMP (Fig. 2a and 2c).Interestingly, in our aforementioned experiments with ATP and goethite, 20-24% of the initial ATP-P remained as AMP-P in solution and no ADP was detected after nearly all the ATP reacted with goethite was transformed or adsorbed (SI, Fig. S10).Thus, this accumulation of AMP in the ATPgoethite experiment can be explained by the lower reactivity of goethite for AMP relative to ATP (Fig. 2c; SI, Fig. S10).When ATP was reacted with hematite, we measured a conversion of 17-23% of the initial ATP-P to ADP-P and 5-7% to AMP-P, consistent with lower reactivity for both ADP and AMP compared to ATP (SI, Fig. S10).Notably, the silicate and aluminosilicate minerals did not exhibit any adsorption or catalytic reactivity towards AMP (Fig. 2c).
In contrast to AMP, all the silicate and aluminosilicate minerals adsorbed phytate (from 5% to 50% of the total reacted phytate-P) and, rather than an Fe oxide, the clay illite adsorbed the most phytate (Fig. 2d).However, similar to the results with AMP and ATP, catalytic reactivity towards phytate was only obtained with Fe oxides, specifically hematite and goethite (Fig. 2d).Relative to controls, phytate-derived particulate Pi was higher by 8 -14% (p < 0.01) with hematite and by 24 -32% with goethite (p < 0.001); the particulate Pi with ferrihydrite, however, corresponded to the adsorption of solution Pi in the control experiment (Fig. 2d).For G6P, some G6P adsorption was observed with two of the aluminosilicates (mica and kaolinite) and two of the Fe oxides (ferrihydrite and goethite) (Fig. 2e).But, as with phytate, the most significant adsorption was with ferrihydrite, with 47 -78% of the reacted G6P found as particulate Porg (Fig. 2e).None of the investigated minerals catalyzed G6P dephosphorylation (Fig. 2e).
In sum, our data revealed that the silicates and aluminosilicates either had minimal to no catalytic reactivity or exhibited some extent of adsorption reactivity.Only the Fe oxides were found to catalyze the dephosphorylation of both phosphomonoester and phosphoanhydride-bearing compounds.Importantly, the Fe oxide-catalyzed reactions seemed to be dependent on both the mineral surface chemistry and the type of Porg species.As proposed previously 14 , we expect the differences in reactivity may stem from the binding conformations of different Porg on the mineral surface.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): I appreciate the authors had processed most of my comments and after major revision, the manuscript looks befter than the inifial version.I am sfill very concerned about use P K-edge XANES to disfinguish P species within different mineral phases and within different P species.In figure 2c and lines 225-230/423-426, the author observed the significant hydrolysis of AMP by ferrihydrite.But with comparison, ferrihydrite showed super limited hydrolysis ability of phytate (Pi in phytate may come ~4% impurity) and G6P, which three P species all contain only one P-O-C bond.Why here ferrihydrite can hydrolyze more than 70% AMP?Why other two Fe oxides show limited AMP hydrolysis ability?what mechanism/surface structure drives such a high amount of AMP's P-O-C cleavage by ferrihydrite, but not for G6P's P-O-C breakage?Baldwin (1995) indicated no such big difference or P-O-C cleavage between different iron oxides and Tong (2020) indicates that the main force to drive C-O-P breakage comes from the exposure facets of crystalline iron oxides.Thus, this data does not make sense, which may come from P K-edge XANES fifting (70% Pi in parficulate phase, but without any release of Pi into solufion, which does not also make sense).I suggest the author should check P K-edge XANES for these AMP-iron oxide samples to get accurate fifting results.Thus, I recommend a minor revision and the author should process this important concern, which might significantly challenge the conclusions here.

Reviewer #1 (Remarks to the Author): To address clearly the comments provided by Reviewer 1, we divided up the one paragraph provided by the reviewer into several comments as detailed below.
Comment 1: I appreciate the authors had processed most of my comments and after major revision, the manuscript looks better than the initial version.

Response:
We thank the reviewer for acknowledging our efforts.It was a herculean effort to secure on very short notice additional beamtime at a synchrotron in Australia during the shutdown of the Stanford synchrotron in California.We have done experiments with additional P org biomolecules with five different minerals, in addition to the experimental data with two other P org compounds that were already presented in the paper.
Comment 2: I am still very concerned about use P K-edge XANES to distinguish P species within different mineral phases and within different P species.
Response: Based on this comment from the reviewer, we would like to clarify that we do not apply the XANES technique beyond its limitation.As pointed out in the Introduction, the P K-edge XANES technique has been demonstrated to distinguish between organic P and inorganic P bound to Fe oxides (see Lines 72-79): "Advances in mineral surface characterization by synchrotron-based P K-edge X-ray absorption near-edge structure (XANES) spectroscopy have made it possible to distinguish between P i and P org bound to Fe in minerals 28 or Fe oxides in a soil matrix 29 .In terms of monitoring mineral-mediated P org recycling, application of the XANES technique with ferrihydrite revealed the generation of particulate P i from adsorbed ribonucleotides, while P i was notably absent from solution 14 .This latter finding, which was confirmed by quantifying the dephosphorylated organic products in the solution by LC-MS 14 , highlights the need to perform quantitative analysis of particulate P i , in addition to dissolved P i , especially for minerals such as Fe oxides with strong adsorption affinity for P i species."Furthermore, we have pointed out in the original version of the manuscript both the powerful insights that can be obtained from the XANES technique and for which analysis this technique can have limitations (see Lines 326-332): "It was not possible to use the P K-edge XANES spectra to distinguish the specific P species associated with the different aluminosilicates nor the specific Fe oxide minerals associated with either particulate P i or particulate P org species on Fe oxides (SI, Fig. S14).Specifically, we were able to employ the LCFs to distinguish P i associated with Ca using apatite as a reference, P (without discriminating between P i or P org ) associated with aluminosilicates, P i associated with Fe in Fe oxides, P org species associated with Fe in Fe oxides, and P i in Fe-P i clusters using vivianite as reference (Fig. 4c, 4d, and 4e)." Therefore, in response to the statement by the reviewer, we did not apply P K-edge XANES to all P species and all minerals.This XANES technique was used to quantify the fraction of particulate P i and particulate P org .This can be done for any type of P org types but we can't distinguish between different P org species present simultaneous in the particulate fraction.Importantly, we employed highresolution mass spectrometry to monitor different organic species (both phosphorylated and dephosphorylated organic species) in solution, from which preliminary quantitation of particulate P i can be determined from performing a mass balance on P. For example, in mineral-mediated analysis of ATP, the following mass balance will provide a preliminary account of particulate P i : (P i, particuate ) = (ADP solution ) + 2(AMP solution ) -(P i,solution ).Using such mass balance, we have highlighted previously that the absence of P i in solution does not mean lack of catalyzed reaction; importantly, the presence of dephosphorylated organics highlight missing P i determined to be particulate P i (Klein et.al., 2019).Response: It is reasonable to expect that different energy would be required to cleave the P-O-C bond in our different investigated phosphomonoesters because the different organic structures attached to the phosphoester bond: a ribonucleoside composed of a sugar attached to heterocycling nitrogenous ring in AMP, versus only a sugar moiety in G6P, versus a inositol in phytate.In fact, Wan  et.al. (2022) reported, based only on monitoring aqueous P i , 10-fold to 25-fold higher P i production from AMP than from G6P and phytate reacted with hematite-these previous experiments were done with 60% less mineral (per mass) in solution and with 20-fold higher concentration for G6P and AMP and 3-fold higher concentration for phytate compared to our experiments.Furthermore, different rates of enzymatic dephosphorylation were reported for these different phosphomonoester P org types (Solhtalab et.al., 2021).Therefore, based on these previous findings, it is not surprising that we found less P i production with G6P compared to AMP reacted with the Fe oxides.
With respect to mechanisms, we would like to point out that the motivation of the current study was to explore whether mineral-mediated catalysis of P org recycling, which was proposed with pure minerals, can actually occur with different natural P org sources, with different minerals, and, importantly, within natural environmental samples obtained from a forest soil and a lake sediment.Now, it is worthwhile for the field to invest in figuring out the mechanisms of this process because we have provided evidence, for the first time, that this mineral-mediated catalysis can occur within environmental samples and, importantly, we obtained abiotic rates that were comparable to enzymatic rates reported in certain soils.Understanding and determining the mechanisms that influence Fe oxide reactivity, which are beyond the scope of the current manuscript, is an important subject for future investigation.We added discussion on exploration of these mechanisms in recently published studies as well as pointing out areas that need to be investigated to gain further insights.(see Lines 452-463): "Our findings corroborate the role of surface chemistry in dictating the extent of catalytic reactivity of the Fe oxide.First, P org dephosphorylation by the Fe oxides was not found to be dependent on surface area.Despite a 14-fold lower surface area for goethite compared to ferrihydrite (16.0 m 2 g -1 versus 230 m 2 g -1 ), goethite was found to be the most reactive, dephosphorylating nearly 7-fold higher amount of the total P org added as ATP.Second, when accounting for the P i binding site density to normalize the rate of dephosphorylation, goethite still exhibited a turnover number up to 9-fold greater than ferrihydrite.Regarding the relevance of other chemical characteristics of the mineral structure, a study with hematic proposed Lewis acid sites and Fe coordination to be determining factors for the extent of hydrolytic cleavage of a synthetic P org by this Fe oxise 15,57 .Our results with naturally-occurring P org showed markedly different reactivity between our three investigated Fe oxides (goethite, ferrihydrite, and hematite) for the same P org compound.Therefore, is Fe oxide-dependent catalytic reactivity highlights the importance of further investigation into the importance of Fe oxide surface chemistry and surface acidity."Response: It is not clear to us what the objection of the Reviewer is because we are reporting our data that were obtained from meticulously performed experiments with independent replicates.It is worthwhile to note that differences in P org dephosphorylation by different Fe oxides has been reported previously by Li et.al. 2020, a reference highlighted by the reviewer (as stated above).Specifically, Li et.al. (2020) reported greater dephosphorylation of pNPP, a synthetic P org , by goethite than by hematite.In agreement with this finding, our results showed no reactivity with hematite but there was reactivity with goethite and ferrihydrite, albeit direct comparison between pNPP and the ribonucleotide AMP is not appropriate (as we already discussed above).
As highlighted both in the abstract and the introduction of our manuscript, the motivation for using XANES technique is because it is critical to consider Fe oxide surfaces as both adsorbents and catalysts whereby generated P i from the mineral catalysis may be trapped on the surface and thus absent from the solution phase.As we have pointed out in the Introduction, a previous study (Klein  et al., 2019) had reported the absence of P i in solution while dephosphorylated organic species were detected in solution by high-resolution LC-MS, which led to the quantification of missing P i bound to ferrihydrite an Fe oxide surface (See our response to Comment 2 above).
Comment 6: I suggest the author should check P K-edge XANES for these AMP-iron oxide samples to get accurate fitting results.Thus, I recommend a minor revision and the author should process this important concern, which might significantly challenge the conclusions here.Response: We were transparent with our XANES data and how they were fitted (see SI Figs S7-S10, Fig. S15, and Table S3, S5-S7) in response to comments during the first round of reviews.It is important to note that the application of the XANES technique has been validated previously, as we have highlighted in the Introduction (also see our response to Comment 2).This technique has been previously documented to distinguish between inorganic P and organic P either bound to pure Fe oxides or found in the environmental matrices (Prietzel et.al. 2016, Eusterhues et.al., 2023).Here we are applying this technique to monitor, for the first time the de novo generation of inorganic P Baldwin, Darren S. "Organic phosphorus in the aquafic environment."Environmental Chemistry 10.6 (2013): 439-454.Baldwin, Darren S., et al. "Phosphate ester hydrolysis facilitated by mineral phases."Environmental science & technology 29.6 (1995): 1706-1709.George, Timothy S., et al. "Organic phosphorus in the terrestrial environment: a perspecfive on the state of the art and future priorifies."Plant and Soil 427 (2018): 191-208.Moore, Oliver W., et al. "Long-term organic carbon preservafion enhanced by iron and manganese."Nature (2023): 1-6.Turner, Benjamin L., et al. "Inositol phosphates in the environment."Philosophical Transacfions of the Royal Society of London.Series B: Biological Sciences 357.1420 (2002): 449-469.Wan, Biao, et al. "Rethinking the biofic and abiofic remineralizafion of complex phosphate molecules in soils and sediments."Science of The Total Environment 833 (2022): 155187.Wan, Biao, et al. "Iron oxides catalyze the hydrolysis of polyphosphate and precipitafion of calcium phosphate minerals."Geochimica et Cosmochimica Acta 305 (2021): 49-65.Wan, Biao, et al. "Manganese oxide catalyzed hydrolysis of polyphosphates."ACS Earth and Space Chemistry 3.11 (2019): 2623-2634.
both C-O-P and P-O-P bonds) a typical organic phosphate in natural environments?Many literatures suggested that, in nature, the main organic P species include phosphate monoester, phosphate diester, and phosphonate (Baldwin,, Environ Chem, 2013; George et al., Plant Soil, 2018).Inositol phosphates are the main organic P storage in terrestrial plants and the most abundant organic P in soils (Turner et al., 2002).How about the catalytic activity of Fe oxides toward the dephosphorylation of phosphate monoester in your soils and sediment incubation?Since they are more abundant Ps in the environment, we cannot neglect their transformation caused by Fe oxides.Wan et al., showed that Fe oxides have low reactivity for C-O-P degradation and high reactivity for P-O-P degradation (Wan et al., Geochim Cosmochim Ac 2021; Sci Total Environ, 2022).If the catalyzed degradation of ATP by Fe oxides might be specified from the cleavage of P-O-P bond, instead of C-O-P bonds, which will weaken your conclusions about organic P (C-O-P bond-including P) mineralization.You may need to consider adding a C-O-P's organic P to see how Fe oxides catalyze purely organic P mineralization and to strengthen your conclusions.
What are the parameters in your soil and sediment samples' porewater?Local temperature?You set up pH 7 and 0.1 M NaNO3 + 0.01 M NaHCO3 to perform experiments.Why not use the porewater conditions to do experiments?Which might be closer to realistic reaction conditions and show the true catalytic reactivity/rates of Fe oxides in organic P mineralization.

Comment 3 :
In figure 2c and lines 225-230/423-426, the author observed the significant hydrolysis of AMP by ferrihydrite.But with comparison, ferrihydrite showed super limited hydrolysis ability of phytate (Pi in phytate may come ~4% impurity) and G6P, which three P species all contain only one P-O-C bond.Why here ferrihydrite can hydrolyze more than 70% AMP?Why other two Fe oxides show limited AMP hydrolysis ability?what mechanism/surface structure drives such a high amount of AMP's P-O-C cleavage by ferrihydrite, but not for G6P's P-O-C breakage?

Comment 4 :
Baldwin (1995) indicated no such big difference or P-O-C cleavage between different iron oxides and Tong (2020) indicates that the main force to drive C-O-P breakage comes from the exposure facets of crystalline iron oxides.Thus, this data does not make sense, which may come from P K-edge XANES fitting (70% Pi in particulate phase, but without any release of Pi into solution, which does not also make sense).References Li, Tong, et al. "Enhanced hydrolysis of p-nitrophenyl phosphate by iron (hydr) oxide nanoparticles: Roles of exposed facets."Science & Technology 54.14 (2020): 8658-8667.Baldwin, Darren S., et al. "Phosphate ester hydrolysis facilitated by mineral phases."Environmental science & technology 29.6 (1995): 1706-1709.